The overall objective of the proposed research is to establish how Ca 2 ion and cAMP interact to regulated differentiation and function of the cytoskeleton of the testicular Sertoli cell using the Sertoli cell-enriched testis and isolated Sertoli cells as model systems. We will first evaluate changes in the synthesis distribution, and assembly of microtubule (tubulin) and microfilaments (actin) together with proteins involved in the action of Ca 2 ion (calmodulin, myosin light chain kinase) and cAMP (Protein kinase inhibitor, catalytic subunit of cAMP-dependent protein kinase). Studies will include localization of the proteins by immunofluorescence and immunocytochemical methods, quantitation of rates of synthesis and degradation as well as steady state levels of the proteins, and determination of mRNA levels by hybridization to complementary cDNA probes. In all instances we will evaluate how the parameters may be altered by Ca 2 ion and cAMP. Next we will investigate how the assembly/disassembly of microtubules is regulated especially in regards to the roles of Ca 2 ion Calmodulin and cAMP. For this aim permiablized Sertoli cells and sperm tail pieces will be used as mucleation sites for 6S tubulin. Changes in enzymes regulated by Ca 2 ion and cAMP will be determined during Sertoli cell differentiation and in response to hormonal stimulation. The enzymes include those involved in glycogen metabolism (glycogen synthase, phosphorylase, phosphorylase kinase) and contractility (myosin light chain kinase). Finally we will evaluate roles for cAMP, Ca 2 ion, protein phosphorylation and the cytoskeleton in regulation of specific protein secretion from isolated Sertoli cells in culture.